In vitro synthesis of lactose permease to probe the mechanism of membrane insertion and folding.

Insertion and folding of polytopic membrane proteins is an important unsolved biological problem. To study this issue, lactose permease, a membrane transport protein from Escherichia coli, is transcribed, translated, and inserted into inside-out membrane vesicles in vitro. The protein is in a native conformation as judged by sensitivity to protease, binding of a monoclonal antibody directed against a conformational epitope, and importantly, by functional assays. By exploiting this system it is possible to express the N-terminal six helices of the permease (N(6)) and probe changes in conformation during insertion into the membrane. Specifically, when N(6) remains attached to the ribosome it is readily extracted from the membrane with urea, whereas after release from the ribosome or translation of additional helices, those polypeptides are not urea extractable. Furthermore, the accessibility of an engineered Factor Xa site to Xa protease is reduced significantly when N(6) is released from the ribosome or more helices are translated. Finally, spontaneous disulfide formation between Cys residues at positions 126 (Helix IV) and 144 (Helix V) is observed when N(6) is released from the ribosome and inserted into the membrane. Moreover, in contrast to full-length permease, N(6) is degraded by FtsH protease in vivo, and N(6) with a single Cys residue at position 148 does not react with N-ethylmaleimide. Taken together, the findings indicate that N(6) remains in a hydrophilic environment until it is released from the ribosome or additional helices are translated and continues to fold into a quasi-native conformation after insertion into the bilayer. Furthermore, there is synergism between N(6) and the C-terminal half of permease during assembly, as opposed to assembly of the two halves as independent domains.